The long term objectives of this project are to acquire a molecular level understanding of the mechanisms that govern cellular control of metabolic pathways. In particular, to study the actual protein molecules involved in the regulatory process, to examine the molecular basis by which an enzyme can regulate its own activity, and to determine how the cell can regulate the biosynthesis of that particular enzyme. Of all the metabolic pathways, the biosynthesis of the purines and pyrimidines, which are required in equal amounts for DNA synthesis, are perhaps the most important Although these pathways have been studied on a macroscopic level, advances in crystallography and biotechnology now make it possible to probe the structure and function of the proteins involved in the control of these pathways at the microscopic level. The understanding of cellular regulation and the proteins involved in the control process will have a great impact on our grasp of cellular differentiation and, as a consequence of this, cures for cancer and birth defects may be found. For the next project period, emphasis will be directed towards two aspects of control of the pyrimidine pathway; first, the enzyme aspartate transcarbamoylase which regulates this pathway by a combination of genetic, metabolic and allosteric control mechanisms; and second, the pyrS gene, the gene-product of which exerts control directly by activating the expression of two of the enzymes of the pathway. The X-ray structures of aspartate transcarbamoylase in the two allosteric forms provides for the first time the necessary structural information to probe this complex system by single amino acid replacements. By the analysis of mutant forms of this enzyme, it will be possible to determine on a functional basis how this enzyme can exert metabolic and allosteric control over pyrimidine biosynthesis. The understanding of this system on the molecular level is particularly important because this system has become a model for the understanding of a diverse number of biological phenomena. The specific aims for this project period are: (1) use the X-ray structures of aspartate transcarbamoylase and functional studies employing site- specific mutants and hybrids to develop and test an integrated model for homotropic cooperativity (allosteric control) and the heterotropic interactions (metabolic control) in this enzyme, (2) determine the molecular level details of the catalytic mechanism of aspartate transcarbamoylase, (3) determine if second sphere interactions are important for catalysis and cooperativity in proteins in general and in aspartate transcarbamoylase in particular, and (4) purify the pyrS gene-product and determine its interaction with the promoters of the pyrimidine pathway.